1. Field of the Invention
This invention relates to novel polynucleotides which encode a mutant human carboxylesterase enzyme, polypeptides encoded by these polynucleotides and vectors and host cells comprising these vectors which express the enzyme. This enzyme is capable of metabolizing chemotherapeutic prodrugs and inactive metabolites into active drug. The instant invention thus relates to compositions comprising these polynucleotides and methods for sensitizing selected cells to a prodrug by transfecting the cells with a polynucleotide placed under the control of a disease-specific responsive promoter. Sensitized cells can then be contacted with a prodrug to alter the activity of the cell in some manner, such as inhibition of tumor cell growth. Alternative methods contemplated by the invention would apply to treatment of drug addiction and resistance to chemical weapons. The invention further includes novel drug screening assays for identifying new prodrugs that are activated by this enzyme.
2. Background of the Invention
Development of effective human anti-cancer drugs has often been hindered by the inability of tested agents to selectively target tumor cells over non-tumor cells. This lack of selectivity of many anti-cancer drugs often leads to unwanted and often serious side effects in patients that can limit the dose of the drug administered. A goal of all human drug therapies, regardless of the disease or condition being treated, is to be able to administer the lowest dose of a drug that produces the desired clinical effect. Therefore, strategies for more efficient drug delivery are continuously sought.
In the case of certain anti-cancer drugs, one strategy has been development of prodrug forms of active compounds that can increase the bioavailability of the drug and increase its ability to effectively kill tumor cells. One example of this prodrug strategy has been the development of carboxylesterase (CE) pro-drugs (Roosebaum, et al. 2004. Pharmacol. Rev. 56:53-102).
CEs are ubiquitous serine esterase enzymes that catalyze conversion of carboxylic esters to an alcohol and a carboxylic acid as well as hydrolyzing amides, thioesters, phosphoric acid esters and acid anhydrides. In some cases, CE enzyme activity is responsible for the detoxification of xenobiotics. CEs are present in high levels in both normal and tumor tissue, especially in liver, kidney, testis, lung and plasma. In addition to their known ability to detoxify certain chemicals, recent research has focused on the ability of these enzymes to be used in design of prodrug forms of certain cytostatic drugs (Roosebaum, et al. 2004. Pharmacol. Rev. 56:53-102). Examples of the application of CE activity to prodrug development are CPT-11, paclitaxel-2-ethylcarbonate, and capecitabine. In all three of these cases, CE activity leads to production of an active cytostatic drug, SN-38, paclitaxel, and 5′-DUFR, respectively. In addition, a particular human CE known as hCE1 has been shown to catalyze the hydrolysis of certain drugs of abuse, specifically heroin and cocaine, and to catalyze the transesterification of cocaine in the presence of ethanol to its toxic metabolite, cocaethylene (Redinbo, et al. 2003. Biochem. Soc. Trans. 31:620-624). hCE1 is also being developed by the United States military as a prophylactic agent for treating potential exposures to chemical weapons such as the organophosphates Sarin, Soman, Tabun, and VX gas (Redinbo, et al. 2003. Biochem. Soc. Trans. 31:620-624).
CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is a prodrug that has been investigated for the treatment of cancer, and is converted to the active drug known as SN-38 (7-ethyl-10-hydroxy-camptothecin) (Tsuji, et al. 1991. J. Pharmacobiol. Dynamics 14:341-349; Satoh, et al. 1994. Biol. Pharm. Bull. 17:662-664). SN-38 is a potent inhibitor of topoisomerase I (Tanizawa, et al. 1994. J. Natl. Cancer Inst. 86:836-842; Kawato, et al. 1991. Cancer Res. 51:4187-4194), an enzyme whose inhibition in cells results in DNA damage and induction of apoptosis (Hsiang, et al. 1989. Cancer Res. 49:5077-5082). In addition to metabolism to SN-38, in humans CPT-11 is also metabolized to a compound known as APC (Haaz, et al. 1998. Cancer Res. 58:468-472). APC has little, if any, anti-tumor activity and is not converted to an active metabolite in humans (Rivory, et al. 1996. Cancer Res. 56:3689-3694). CPT-11 has demonstrated remarkable anti-tumor activity in pre-clinical models and Phase I/II clinical trials (Furman, et al. 1999. J. Clin. Oncol. 17:1815-1824; Houghton, et al. 1996. Clin. Cancer Res. 2:107-118; Houghton, et al. 1995. Cancer Hcemother. Pharmacol. 36:393-403), and as such is being tested against a variety of human malignancies. However, myelosuppression and secretory diarrhea limit the amount of drug that can be administered to patients. Accordingly, before this promising anti-cancer agent can be used successfully, these dose-limiting toxicities must be overcome. CPT-11 is currently approved for use in human colon cancer.
The active anti-tumor agent of CPT-11, SN-38, can be detected in the plasma of animals and humans minutes after the administration of CPT-11 (Stewart, et al. 1997. Cancer Chemother. Pharmacol. 40:259-265; Kaneda, et al. 1990. Cancer Res. 50:1715-1720; Rowinsky, et al. 1994. Cancer Res. 54:427-436), suggesting that a CE enzyme present in either serum or tissues can convert the camptothecin analog to its active metabolite. When CPT-11 is administered to humans, typically less than 5% of the drug is converted to SN-38, which is in contrast to mice where greater than 50% of the drug is hydrolyzed to SN-38 within the first hour of dosing (Morton, et al. 2005. Cancer Chemother. Pharmacol. 56:629-636). This may be due to either the different levels of CEs expressed in these species, or the proficiency of drug hydrolysis of the different CEs.
Since the activation of CPT-11 in humans is relatively inefficient, CE enzyme prodrug therapy approaches have been examined. For example, an enzyme/prodrug therapy approach using a rabbit liver CE (rCE) which is much more efficient at drug activation has been developed (Danks, et al. 1999. Cancer Res. 5:917-924; Meck, et al. 2001. Cancer Res. 61:5083-5089; Potter, et al. 1998. Cancer Res. 52:2646-2651; Wagner, et al. 2002. Cancer Res. 62:5001-5007; Wierdl, et al. 2001. Cancer Res. 61:5078-5082). Using the rCE in therapy, increased sensitivity to CPT-11 was accomplished in human tumor cells grown in culture and in xenografts in immune-deprived mice. It has been suggested, however, that the application of the rCE to human therapy may be limited due to the potential immunogenicity of the lagomorph protein. Human CE enzymes have been examined, but, in vitro studies suggest that human intestinal CE (hiCE) is not as efficient at drug activation when compared to rCE (Humerickhouse, et al. 2000. Cancer Res. 60:1189-1192; Khanna, et al. 2000. Cancer Res. 60:4725-4728). Additionally, while sensitization of cells to CPT-11 expressing hiCE has been reported (e.g., Khanna, et al. 2000. Cancer Res. 60:4725-4728), studies indicate that the levels and duration of hiCE expressed are much lower than can be achieved with rCE.
The development of new effective treatment strategies for cancer is dependent upon the availability of specific drug screening assays. Specific drug screening assays can involve isolated target tissue models, i.e., isolated heart, ileum, vasculature, or liver from animals such as rabbits, rats, and guinea pigs, wherein the target tissue is removed from the animal and a selected activity of that target tissue is measured both before and after exposure to the candidate drug. An example of a selected activity measured in drug screening assays to identify new cancer agents is the activity of enzymes such as topoisomerase I or II, which are known to modulate cell death. Such assays can also be used to screen for potential prodrugs which are converted to the active metabolite in selected tissues or to identify selected tissues capable of converting prodrug to its active metabolite.
However, any molecular event that is shown to be modified by a novel class of compounds can be developed as a screening assay for selection of the most promising compounds for therapeutic development. In fact, the idea of modulating cells at the genomic level has been applied to the treatment of diseases such as cancer. Gene therapy for treatment of cancer has been the focus of multiple clinical trials approved by the National Institutes of Health Recombinant DNA Advisory Committee, many of which have demonstrated successful clinical application (Hanania, et al. 1995. Am. Jour. Med. 99:537-552; Johnson, et al. 1995. J. Am. Acad. Derm. 32(5):689-707; Barnes, et al. 1997. Obstetrics and Gynecology 89:145-155; Davis, et al. 1996. Current Opinion in Oncology 8:499-508; Roth and Cristiano 1997. J. Natl. Canc. Inst. 89(1):21-39). To specifically target malignant cells and spare normal tissue, cancer gene therapies must combine selective gene delivery with specific gene expression, specific gene product activity, and, possibly, specific drug activation. Significant progress has been made in recent years using both viral (retrovirus, adenovirus, adeno-associated virus) and nonviral (liposomes, gene gun, injection) methods to efficiently deliver DNA to tumor sites. Genes can be transfected into cells by physical means such as scrape loading or ballistic penetration, by chemical means such as coprecipitation of DNA with calcium phosphate or liposomal encapsulation; or by electro-physiological means such as electroporation. The most widely used methods, however, involve transduction of genes by means of recombinant viruses, taking advantage of the relative efficiency of viral infection processes. Current methods of gene therapy involve infection of organisms with replication-deficient recombinant viruses containing the desired gene. The replication-deficient viruses most commonly used include retroviruses, adenoviruses, adeno-associated viruses, lentiviruses and herpes viruses. The efficacy of viral-mediated gene transfer can approach 100%, enabling the potential use of these viruses for the transduction of cells in vivo.
Adenovirus vector systems in particular have several advantages. These include the fact that non-dividing cells can be transduced; transduced DNA does not integrate into host cell DNA, thereby negating insertional mutagenesis; the design of adenoviral vectors allows up to 7 kb of foreign DNA to be incorporated into the viral genome; very high viral titers can be achieved and stored without loss of infectivity; and appropriate plasmids and packaging cell lines are available for the rapid generation of infectious, replication-deficient virus (Yang 1992. Crit. Rev. Biotechnol. 12:335-356). The effectiveness of adenoviral-mediated delivery of genes into mammalian cells in culture and in animals has been demonstrated.
To increase the specificity and safety of gene therapy for treatment of cancer, expression of the therapeutic gene within the target tissue must also be tightly controlled. For tumor treatment, targeted gene expression has been analyzed using tissue-specific promoters such as breast, prostate and melanoma specific promoters and tumor-specific responsive promoters such as carcinoembryonic antigen, HER-2/neu, Myc-Max response elements, DF3/MUC (Dachs, et al. 1997. Oncol. Res. 9:313-25). For example, the utility of herpes simplex virus thymidine kinase (HSV-TK) gene ligated with four repeats of the Myc-Max response element, CACGTG, as a gene therapy agent for treatment of lung cancer with ganciclovir was examined in c-, L- or N-myc-overexpressing small cell lung cancer (SCLC) cell lines (Kumagai, et al. 1996. Cancer Res. 56(2):354-358). Transduction of the HSV-TK gene ligated to this CACGTG core rendered individual clones of all three SCLC lines more sensitive to ganciclovir than parental cells in vitro, thus suggesting that a CACGTG-driven HSV-TK gene may be useful for the treatment of SCLC overexpressing any type of myc family oncogene. Additional experiments with c-myc have focused on the use of the ornithine decarboxylase (ODC) promoter gene. Within the first intron of the ODC gene are two CACGTG “E boxes” that provide binding sites for the c-myc protein when bound to its partner protein known as max. Mutation of the E box sequence results in the inability of c-myc to transactivate the ODC promoter. Previous reports indicate that reporter constructs containing the ODC promoter fused upstream of the chloramphenicol acetyltransferase gene immediately adjacent to the second exon were activated in cells that overexpress c-myc (Bello-Fernandez, et al. 1993. Proc. Natl. Acad. Sci. USA 90:7804-7808). In contrast, transient transfection of promoter constructs in which the E boxes were mutated (CACGTG to CACCTG) demonstrate significantly lower reporter gene activity. These data suggest that it is possible to activate transcription of specific genes under control of the c-myc responsive ODC promoter. In the case of N-myc, N-myc protein is a basic helix-loop-helix (BHLH) protein that can dimerize with proteins of the same class. N-myc dimerizes with the BHLH protein max to form a complex that binds to the CACGTG motif present in gene promoters, such as ODC, resulting in transactivation and expression of specific genes containing this sequence (Lutz, et al. 1996. Oncogene 13:803-812). Studies in a neuroblastoma cell line and tumors have shown that binding of N-myc to its consensus DNA binding sequence correlates with N-myc expression, data that indicate that the level of N-myc in neuroblastoma cells is a determining factor in expression of proteins under control of promoters containing the CACGTG sequence (Raschella, et al. 1994. Cancer Res. 54:2251-2255). Inhibition of expression of the c-myc gene via antisense oligonucleotides as a means for inhibiting tumor growth has also been disclosed (Kawasaki, et al. 1996. Artif. Organs 20(8):836-48).
In the present invention, a mutant human CE has been developed that can activate CPT-11 as efficiently as CE from rabbit liver (rCE). Polynucleotides encoding this mutated human carboxylesterase enzyme or active fragments thereof and polypeptides encoded thereby which are capable of metabolizing the chemotherapeutic prodrug CPT-11 to active drug SN-38 are disclosed. This mutant human carboxylesterase enzyme also has potential applications to treatments involving other potential substrates such as certain drugs of abuse (e.g., cocaine and heroin) and certain types of chemical weapons (e.g., organophosphates). It has also been found that compositions comprising a polynucleotide of the present invention and a disease-specific responsive promoter can be delivered to selected tumor cells to sensitize the tumor cells to the chemotherapeutic prodrug CPT-11, thereby inhibiting tumor cell growth.